A 700 kDa protease called the 20S proteasome (20S) is essential for eukaryotic life. Isolated 20S are inactive because the gate to their internal catalytic chamber is closed. Substrates/products enter/exit the 20S interior via activator complexes that bind to the 20S ends and open the entrance gate. Three classes of activator have been identified: 11S, Blm10, and 19S. Previously we focused on 11S activators and determined 1.1 MDa, 42 subunit structures of 11S complexes with both yeast and archaeal 20S. Remarkably, the archaeal 20S complex crystals diffracted to better than 2.0 A resolution. Based upon these structures, we proposed mechanisms of binding and gate opening, and confirmed these models through mutagenesis, biochemistry, and structure determination of mutant 20S:11S complexes. We further suggested that 19S employs a similar mechanism of binding and stabilizes the same open conformation, and we validated these proposals with mutagenic and biochemical studies. Building on this progress, our future studies seek a detailed understanding of all three classes of proteasome activator. Aim 1 - Blm10. Preliminary data include development of expression and purification protocols that yield active Blm10-20S complex, clarification of data from yeast genetics, determination of an 18 A structure by electron cryomicroscopy, crystallization, and preliminary crystal structure determination at 3.5 A resolution. The emerging structure reveals an extensive contact surface and disordering of the 20S gate to a partially open conformation. An unexpected bonus is that the structure suggests a detailed model for how 19S activator binds and opens the 20S gate. Aim 2 - 11S. Our well-defined PA26:20S system is being exploited to design chimeric constructs that will provide detailed models of how the highly diverged mammalian 11S activators bind and activate proteasomes. This approach will also advance detailed models of how 19S/PAN activator subunits bind and open the proteasome gate. Preliminary crystals have been prepared. Aim 3 - S19. Preparations of the 900 kDa multisubunit 19S activator and its 2.5 MDa complex with 20S (the 26S proteasome) suffer from heterogeneity and are therefore being approached by recombinant expression and reconstitution of functionally significant subcomplexes. Several subcomplexes have been prepared, one of which has yielded preliminary crystals. PUBLIC HEALTH RELEVANCE: Proteasomes are large macromolecular assemblies that are essential in eukaryotes and are attractive targets for the development of new therapeutics, especially for the treatment of cancers. We aim to advance understanding of proteasome regulation through biochemical, genetic, and structural studies of their biological activators.